This proposal outlines a research effort to delineate the structural determinants of catalytic efficiency; inhibitor binding; and metal binding and reactivity in the zinc metalloenzyme, human carbonic anhydrase II (CA II). These principles will then be tested and expanded by redesigning the functional repertoire of the active sites of both CA II and the zinc metalloprotease, carboxypeptidase. Catalytic efficiency and inhibitor potency will be related to protein structure through a combination of mutagenesis, spectroscopy, kinetic analysis, structure determination and theoretical calculations. Experiments are designed to: 1) examine the substrate specificity of CA II; 2) delineate the role of protein ligands in metal binding, specificity and reactivity; 3) define the structural and functional role of the protein context of the metal coordination polyhedron, including second shell hydrogen bonds and a hydrophobic shell; 4) investigate the role of hydrogen bonds to the zinc-solvent ligand in determining the catalytic mechanism; 5) convert the zinc binding site in CA II into a blue copper site and a hydrolytic site similar to that of the zinc proteases; 6) redesign the active site of carboxypeptidase I to catalyze CO2 hydration; 7) characterize the biochemical properties of a physiological plasma inhibitor of CA II, including binding interactions with CA II, similarity to transferrin and primary sequence; and 8) identification of inhibitors specific for other isozymes. Information gained will impact on our understanding of the regulation and physiological importance of carbonic anhydrase isozymes and in the design of isozyme specific inhibitors. Homozygous CA II deficiency in humans has clinical consequences for bone, kidney and brain causing osteopetrosis, renal tubular acidosis and cerebral calcification, respectively. In addition, CA II is potently inhibited by sulfonamides which are used clinically to treat glaucoma. Dissection of structural motifs in CA II and CPA essential for stability and reactivity will increase our understanding of the catalytic mechanism of these and other metalloenzymes, as well as lead to substantial improvement in our understanding of the guiding principles for protein engineering of metal binding sites and designing active site inhibitors. Zinc proteins play indispensable roles in metabolism and gene expression and catalyze crucial physiological reactions such as DNA and RNA polymerization, CO2 hydration, connective tissue and protein degradation, and intermediary metabolism. Malfunction and inhibition of these enzymes has implications for cancer, aging, metabolic diseases, arthritis, immunodeficiency and glaucoma.